Synthesis of [11C]SSR149415 and preliminary imaging studies using positron emission tomography

Article abstract of DOI:10.1016/j.bmcl.2010.03.108

SSR149415 was the first non-peptide vasopressin-(V_1b) receptor antagonist reported. It has been used to probe the role of V_1b receptors in animal models of depression, aggression, and stress-anxiety, and was progressed to clinical tr

Full text of DOI:10.1016/j.bmcl.2010.03.108

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3103–3106
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of [11C]SSR149415 and preliminary imaging studies using
positron emission tomography
Matthias Schönberger a,b, Carmine Leggett c, Sung Won Kim a, Jacob M. Hooker a,d,e,
*
a Brookhaven National Laboratory, Medical Department, Bldg 555, Upton, NY 11973, USA
b Johannes Gutenberg University Mainz, Nuclear Chemistry Department, Fritz Strassmann-Weg 2, 55128 Mainz, Germany
c University of Louisville School of Medicine, Department of Pharmacology and Toxicology, Louisville, KY 40202, USA
d Athinoula A. Martinos Center for Biomedical Imaging, Charlestown, MA 02129, USA
e Division of Nuclear Medicine and Molecular Imaging, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
SSR149415 was the first non-peptide vasopressin-(V1b) receptor antagonist reported. It has been used to
probe the role of V1b receptors in animal models of depression, aggression, and stress-anxiety, and was
progressed to clinical trials for the treatment of depression. Due to the interest in V1b receptors as a ther-
apeutic target and the growing use of SSR149415 in preclinical research, we developed a method to label
SSR145419 with carbon-11 and have studied its pharmacokinetics in non-human primates using positron
emission tomography.
Received 8 March 2010
Revised 26 March 2010
Accepted 26 March 2010
Available online 2 April 2010
Keywords:
Positron emission tomography
Vasopressin
Ó 2010 Elsevier Ltd. All rights reserved.
SSR149415
Carbon-11
OH
Arginine vasopressin (AVP) is a cyclic disulfide peptide hormone
that is synthesized in the hypothalamus, stored in the pituitary, and
secreted into the blood.1 AVP engages several targets including the
three unique AVP receptors (V1a, V1b, and V2) as well as oxytocin
receptors. Elevation of AVP in the bloodstream causes antidiuresis
and moderate vasoconstriction mediated predominantly by V1a
and V2 receptors and their downstream signaling pathways. V1b
receptors (also known as V3 receptors) are distributed in the central
nervous system and modulate release of the stress hormone ACTH
in response to AVP.2 Due to this function, V1b receptors have been
implicated as targets for exploring and potentially treating stress-
related disorders such as anxiety and depression.3
Disclosure of the first non-peptide antagonist of V1b, SSR149415,
has facilitated research aimed at improving our understanding of
the physiological role that V1b receptors play in stress-related
disorders.4 SSR149415 exhibits low nanomolar inhibition of V1b
receptors and 100–1000-fold selectivity over V1a, V2, and oxytocin
receptors. Behavioral pharmacology experiments using SSR149415
have demonstrated its anxiolytic- and antidepressive-like effects
as well as its ability to reduce offensive aggression.5 Furthermore,
the use of [3H]SSR149415 for in vitro saturation and autoradiogra-
phy experiments indicated an interesting binding profile in certain
tissues including the pituitary.6
O
N
Cl
O
N
N
O
O
S O
O
O
V1b Antagonist
SSR149415
Given the interest in V1b receptor pharmacology and the use of
SSR149415 as a therapeutic drug7 we developed a method to label
SSR149415 with carbon-11 to facilitate evaluation of its pharmaco-
kinetics in vivo using positron emission tomography (PET)—a pow-
erful tool for understand drug behavior.8 Herein, we describe a
route for the synthesis of SSR149415, which was adapted from pat-
ent literature9. This is the first full report of its synthesis, isolation,
and characterization data. In addition, we detail our strategy and
method to label SSR149415 with carbon-11. Using this strategy
we have performed initial in vivo characterization in non-human
primates using PET, which indicate that SSR149415 has low blood
brain barrier (BBB) penetration and brain uptake.
* Corresponding author.
E-mail address: hooker@nmr.mgh.harvard.edu (J.M. Hooker).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.03.108

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M. Schönberger et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3103–3106
O
Cl
O
O
N
H
OH
OCH3
Br
OCH3
Li
3
Cl
i
iii
O
ii
N
H
1
2
4
OH
OH
O
7
O
O
iv
Cl
N
N
Cl
Cl
Cl
O
O
H
N
O
O
O
O
N
H
N
N
N
N
H
N
H
5
6
8
OH
Scheme 1. Synthesis of SSR149415 part 1. Addition of the anisole- and proline derivative to the isatine‘s carbonyl C-3. Reagents and conditions: (i) nBuLi, THF, À78 °C,
10 min; (ii) THF, À78 °C to rt, 30 min, 49%; (iii) SOCl2, Pyr, DCM, 0 °C to rt, 30 min; (iv) TEA, DCM, 0 °C to rt, 48 h, 40% (+/-).
Cl
S
Cl
S
O
O
O
O
OH
O
O
O
i
ii
+
OH
9
O
O
O
10
11
12
Scheme 2. Functionalization of resorcinol. Reagents and conditions: (i) Allyl bromide, CH3I, K2CO3, acetone, rt to 80 °C, 6 h, 80%; (ii) (1) PCl5, DCM, 0 °C; (2) SO3/H2SO4, 5 min,
24% (mxt 11/12).
OH
O
R1
R2
Cl
S
N
OH
Cl
O
8
O
O
O
O
S O
CH3
CH3
CH3
OR1
N
13
14
i
N
N
+
CH2CHCH2
Cl
O
OR1
O
O
CH3
CH2CHCH2
N
15
OR2
N
H
OR2
Scheme 3. Synthesis of SSR149415 part 2 and precursor preparation. Reagents and conditions: (i) NaH, DMF, 0 °C, 3 min, quant.
The synthetic procedure for both SSR149415 and a precursor for
carbon-11 labeling, was based on additions of pendant groups to a
commercially available isatin core, Scheme 1. Although asymmet-
ric additions to isatins are known10 we chose to perform a racemic
synthesis of alcohol 4, relying on the separation of diastereomers
formed in subsequent steps with achiral techniques. To avoid pro-
tection of the amide nitrogen in the first step of our scheme, we
employed an excess of the aryl lithiate 2 formed from o-bromoani-
sole. The reaction of 2 with 3 occurred quickly at À78 °C and affor-
ded the racemic mixture of 4 in 49% combined yield.
moval of the excess thionyl chloride, intermediate 5 was treated
with triethylamine and proline fragment 6. The resulting epi-
mers 7 and 8 were separated by flash chromatography (the iden-
tity of the two epimers was later assigned based on optical
rotation data following sulfonamide formation and comparison
to the Sanofi patent).9
To obtain a desmethyl derivative of SSR149415 for use as a car-
bon-11 precursor, an allyl protected sulfonyl chloride building
block was synthesized starting from resorcinol (9), Scheme 2.
Diether 10 was formed in a one pot from a mixed alkylation reac-
tion using methyl iodide and allyl bromide. Following isolation,
compound 10 was treated with excess PCl5 and fuming sulfuric
acid at 0 °C. As expected, both regioisomers 11 and 12 were
obtained. Although they could be separated, we found that it was
more efficient to carry on the mixture of 11 and 12 and perform
the separation during the last step of the precursor synthesis.
Sulfonamide formation at the ‘isatin’ nitrogen 8 was accom-
plished with commercially available 2,4-dimethoxybenzene sulfo-
nylchloride and the mixture of 11 and 12, Scheme 3. Using this
The proline fragment of SS9149415 was derived from commer-
cially available trans-4-hydroxy-L-proline in three steps: protection
of the proline nitrogen as a benzyl carbamate, formation of the
dimethyl amide, and deprotection (see Supplementary data for
details). Using this procedure building block 6 was isolated after
three steps without chromatography proceeding in 85% overall
yield.
To add the proline and isatin fragments, tertiary alcohol 4
was converted to chloride 5 using thionyl chloride. Following re-

M. Schönberger et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3103–3106
3105
Table 1
Characterization of compounds 7, 8, 13 and 16 with chromatography and optical
rotation measurements (see Supplementary data)
7
8
13
16
9.96
RT
Rf
8.99
0.50
7.89
0.42
10.18
0.53
0.51
a2D5
—
—
À0.140
+0.079
Entry 1: Retention times (RT) by HPLC. Entry 2: Rf-values (TLC). Entry 3: Optical
rotation, c = 1 mg/mL (CHCl3), b = 1 cm.
(-) 13
s
s
d
d
d
t
d
d
d
Figure 3. Summed PET images from baboon experiment with [11C]SSR149415. A:
lung, transverse view, (50–420 s), (B) heart muscle, transverse view, (50–420 s), (C)
spine and liver, sagittal view, (50–420 s), (D) liver and gall bladder, coronal view
(50–1200 s). (E) Whole body PET scan of a baboon performed after a 90 min
dynamic scan. (F) transverse, (G) sagittal and (H) coronal view on brain images
centered on pituitary (pt).
(+) 16
s
s
d
d
d
t
d
8.0
7.5
7.0
6.5
ppm
14 and 15 was dissolved in DCM and treated with a catalytic
amount of Pd(PPh3)4 followed by dimethylamine. This led to quan-
titative deprotection after only 10 min at room temperature. The
two regioisomers were separated by preparative reversed phase
chromatography. With both regioisomers in hand, we investigated
methylation (to form SSR149415) and quickly determined that reg-
ioisomer 17 was superior in terms of both labeling yields and ease
of purification by chromatography.
Figure 1. Comparison of SSR149415 (13) and its epimer (16) of opposite optical
rotation using 1H NMR. The aromatic protons can be used for identification.
strategy we obtained SSR149415 (13) and two allyl protected pre-
cursors for carbon-11 labeling (14 and 15). It is worth noting that
we also performed these reactions with compound 7 (the epimer of
8) in order to get full characterization of the epimer of SSR149415
(16). The pharmacologically active epimer was previously assigned
as the one with a negative optical rotation. Since other labs may
want to prepare SSR149415, we have provided Table 1 that sum-
marizes some of the key chromatography and optical rotation
information.
Once we had assigned all of the structures based on the patent
literature, we found that the most facile technique to identify each
epimer was by proton NMR. Figure 1 highlights signals of the aro-
matic protons showing further separated doublets downfield from
the solvent peak and a reversed order of singlet and triplet upfield.
This data should help facilitate quick identification of SSR149415
for those preparing it in the future.
After a brief period of optimizing the reaction of 17 with
[
11C]methyl iodide, we found that the highest yielding reactions
were realized when the supernatant of a saturated NaOH/DMSO
solution was used as solvent/base system at high temperatures
(150 °C) and short reaction times (4 min) (see Supplementary data
for optimized procedure). Figure 2B provides a representative
chromatogram using a radioactivity detector. The radioactive
product eluting at the expected SSR149415 retention time was col-
lected, concentrated and analyzed by a variety of chromatography
experiments for quality control. We also verified the identity of the
peak using LC–MS performing analogous reactions with
[
13C]methyl iodide.
Following our optimized procedure, we were able to synthesize,
isolate, and formulate [11C]SSR149415 in ꢀ40 min from the end of
Palladium-mediated deallyation of a mixture of 14 and 15 was
used to reveal phenol precursors for carbon-11 labeling experi-
ments, Figure 2A (compound 15 is shown singularly for clarity).
To accomplish the deprotection the mixture of allyl-aryl ethers
cyclotron bombardment. A typical preparative run produced 3–
6 mCi (>2 Ci/lmol) of the final product in injectable saline with
10% EtOH after filtration through a 0.22
lM sterile filter.
OH
OH
OH
O
O
O
11
N
N
N
[
C]SSR149415
Cl
Cl
Cl
O
O
O
N
i
ii
N
N
N
S
N
S
N
O
O
O
O
O
O
O
O S O
O
O
O
O
OH
O11CH3
[
11C]-SSR149415
0
5
10
15
20
25
15
17
time (min)
Figure 2. (A) Deallylation revealing precursor and 11C-labeling. (i) Pd(PPh3)4, DMA, DCM, rt, 10 min, quant.; (ii) [11C]CH3I, NaOH/DMSO, 150 °C, 4 min. (B) Semipreperative
HPLC, radioactive trace (see Supplementary data for LC–MS analysis).

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M. Schönberger et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3103–3106
Using [11C]SSR149415 we determined its lipophilicity (log D)
Acknowledgments
and propensity for plasma protein binding (PPB). The log D value
of [11C]SSR149415 received from a series of octanol/water distribu-
tions was 3.22 0.67 (n = 16)—well within the accepted log D val-
ues for passive blood brain barrier penetration of 2.0–3.5.11 In PPB
assays, we determined that only 2.54% 0.43% (n = 4) of the com-
pound was in the free fraction of plasma indicating a strong affinity
for plasma proteins. The nature or specificity of this interaction
was not determined.
This study was supported by Brookhaven National Laboratory
under contract DE-AC02-98CH10886 with the U.S. Department of
Energy, supported by its Office of Biological and Environmental Re-
search. Funding for M.S. was provided by the Study Foundation of
the German People. The authors are grateful to Dr. Michael Schuel-
ler for cyclotron operation and the PET radiochemistry, the imaging
team at BNL (Youwen Xu, Colleen Shea, Lisa Muench, David Alexoff,
Pauline Carter, Payton King, and Don Warner) for carrying out pri-
mate imaging experiments, and to Dr. Joanna Fowler for scientific
input and guidance. M.S. acknowledges Prof. Dr. Frank Rösch for
advice and support during this work.
Several PET imaging experiments were performed using non-
human primates to evaluate the pharmacokinetics and metabolism
of [11C]SSR149415. The average dose for the PET imaging experi-
ments using baboons was 2.5 0.8 mCi [11C]SSR149415, which
was administered intravenously. Dynamic brain images were col-
lected for 90 min. Following this, the animal position was moved
placing the torso in the PET gantry and another injection of
Supplementary data
[
11C]SSR149415 was performed (>2 h after the initial injection).
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.03.108.
Again a 90 min dynamic acquisition was collected. In addition to
these studies, following the final dynamic imaging session, a whole
body scan was recorded to determine the gross distribution of
radioactivity in the animal at the end of the 90 min acquisition.
Starting immediately at the time of injection, blood samples were
taken over the course of 60 min and analyzed by HPLC to deter-
mine the fraction of carbon-11 which was present as parent com-
pound. Interestingly, after approximately 10 min, the drug was
already metabolized to 50%. The entire imaging session was
repeated in a second animal.
Summed-images from these experiments are provided in Figure
3 (time-activity curves and metabolism data can be found in the
Supplementary data).
Using PET, we determined that SSR149415 and/or its radiola-
beled metabolites accumulate predominantly in the liver (0.1–
0.25% ID/cc). We observed low yet noticeable accumulation in
the heart, spine and lung. These were on the order of 0.01–0.03%
ID/cc at their maximum.
References and notes
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Interestingly, we observed very minimal uptake of radioactivity
in the non-human primate brain. We surmise that mechanism of
action of SSR149415 in behavioral pharmacology experiments is
likely not due to interaction of V1b receptors in the brain. However,
we did observe a high concentration of radioactivity in the
pituitary, which has been reported to possess a high density of
V1b-receptors.12 The saturability of the accumulated radioactivity
that we observed has not yet been determined so at this point
we cannot comment on whether the binding is specific.
In conclusion, we have developed a method for the synthesis of
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[
11C]SSR149415 and characterized its pharmacokinetic behavior in
non-human primates. These data should provide valuable insight
into the pharmacological effects that have been observed in behav-
ioral assays using SSR149415.
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